Fermentative production of n-butylacrylate using alcohol acyl transferase enzymes

Abstract

Provided herein is a recombinant nucleic acid molecule, a recombinant microorganism, and a method for fermentative production of n-butylacrylate and other esters from alcohols and acyl-CoA units using alcohol acyl transferase enzymes.

We claim: 
     
       1. A method for fermentative production of n-butylacrylate (n-BA) comprising the steps of:
 i) providing a recombinant microorganism comprising a butanol producing pathway and an acryloyl-CoA producing pathway and expressing an alcohol acyl transferase (AAT) gene encoding an AAT enzyme having an n-BA forming activity; 
 ii) culturing said microorganism under conditions that allow for the production of n-BA; and 
 iii) recovering n-BA from a fermentation broth; 
 wherein the AAT gene encoding an AAT enzyme having an n-BA forming activity is selected from the group consisting of: 
 (I) a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13; 
 (II) a nucleic acid molecule having at least 95% identity to a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13; 
 (III) a nucleic acid molecule hybridizing to the full complement of a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO: 1, 5, 7, 9, 11 and 13; 
 (IV) a nucleic acid molecule encoding a polypeptide selected from the group consisting of SEQ ID NO: 2, 6, 8, 10, 12, and 14; and 
 (V) a nucleic acid molecule encoding a polypeptide having at least 95% identity to a polypeptide selected from the group consisting of SEQ ID NO: 2, 6, 8, 10, 12, and 14. 
 
     
     
       2. A process for fermentative production of n-BA comprising the steps of:
 I) growing in a fermenter a recombinant microorganism comprising a nucleic acid molecule encoding an AAT gene encoding an AAT enzyme having an n-butylacrylate (n-BA) forming activity, wherein the microorganism also comprises a butanol producing pathway and an acryloyl-CoA producing pathway, and wherein the microorganism has an introduced, increased, or enhanced n-butylacrylate forming activity and/or expression of said AAT enzyme; 
 wherein the AAT gene encoding an AAT enzyme having an n-BA forming activity is selected from the group consisting of: 
 (I) a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13; 
 (II) a nucleic acid molecule having at least 95% identity to a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11 and 13; 
 (III) a nucleic acid molecule hybridizing to the full complement of a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO: 1, 5, 7, 9, 11 and 13; 
 (IV) a nucleic acid molecule encoding a polypeptide selected from the group consisting of SEQ ID NO: 2, 6, 8, 10, 12, and 14; and 
 (V) a nucleic acid molecule encoding a polypeptide having at least 95% identity to a polypeptide selected from the group consisting of SEQ ID NO: 2, 6, 8, 10, 12, and 14; and 
 II) recovering n-BA from a fermentation broth obtained in step I). 
 
     
     
       3. The method of  claim 1 , wherein the nucleic acid molecule hybridizing to the full complement of a nucleic acid molecule having the sequence of SEQ ID NO: 1, 5, 7, 9, 11 and 13 hybridizes under high stringency conditions of hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 65° C. 
     
     
       4. The process of  claim 2 , wherein the nucleic acid molecule hybridizing to the full complement of a nucleic acid molecule having the sequence of SEQ ID NO: 1, 5, 7, 9, 11 and 13 hybridizes under high stringency conditions of hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 65° C.